Insofar as an in vitro gene manipulation technology is concerned, it is required that a vector which is accepted by a host be available to allow a foreign DNA to migrate in a cell of the host and to allow the foreign DNA to express the genetic information thereof in the cell of the host. The behavior of a vector is clear, to a considerable extent, for the host-vector system in which Escherichia coli is employed, because efforts have been concentrated thereon so far. At present, however, many efforts are being used for development of host-vector systems which employ various microorganisms other than E. coli, such as Bacillus subtilis which is one of the microorganisms useful from the industrial viewpoint, Actinomycetes which are microorganisms capable of producing antibiotics, and yeast which is widely employed for brewing.
The fundamental requirements for a vector are that the vector has a gene arrangement which is necessary for replication and that the vector has a recognition and cleavage site for a restriction enzyme at which a foreign DNA is inserted. From the practical viewpoint, however, various additional items tabulated below are required for a vector:
1. Each of the specific restriction enzymes which can be employed for a specific gene manipulation has a recognition and cleavage site which is convenient in later steps in the specific gene manipulation process.
2. The vector has a high efficiency in expression of genetic information.
3. The vector has a marker gene which is necessary for detection of a transformant.
4. The vector is readily accepted by a specific host, and the vector has a convenient host range.
5. The vector is stable in the specific host without incurring disturbance.
6. The vector allows a biological confinement which is aimed at reduction of possibilities of any potential biological hazards.
This is the reason why only a few successful cases have been reported for the practically useful vectors even within the category of the host-vector systems employing E. coli or B. subtilis.
In view of the aforementioned facts, we have been using our best efforts for acquisition of plasmid vectors useful for the ultimate purposes of developing host-vector systems which employ E. coli, the biological properties of which are well known and B. subtilis, which is useful for the industrial purpose of producing amylase et al. As a result, applicants were successful in development of a variety of chimeric plasmids which are synthesized from a plasmid retained by Streptococcus faecalis, pAM.alpha.1, and a vector applicable to E. coli, pACYC177, and which are excellent vectors for gene manipulation employing E. coli or B. subtilis.
Applicants have further put forth efforts to develop more useful plasmid vectors. These continued efforts have now resulted in development of excellent chimeric shuttle vectors which not only have beneficial microbial properties comparable to those of known plasmid vectors but also permit of the use of various restriction enzymes. The present invention is particularly concerned with these novel, useful vector.